: The overall objective of this proposal is to define the cellular mechanisms responsible for the malignant transformation of biliary epithelia. Since chronic inflammation of the biliary tree predisposes to malignant transformation of cholangiocytes, the long-term goal is to understand the mechanisms by which inflammatory mediators, especially the inducible form of nitric oxide synthase (iNOS), contributes to the development and progression of biliary tract cancers. The central hypothesis is that NO derived from iNOS expression in cholangiocytes promotes the development and progression of cholangiocarcinoma by inhibiting DNA repair and cellular apoptosis via nitrosylation of critical proteins. Using molecular, biochemical, and cell biological approaches, the PI will test this central hypothesis in three specific aims. The first specific aim is to test the hypothesis that NO directly inhibits base excision DNA repair proteins, especially 8-oxodeoxyguanine glycosylase (hOGG1), resulting in potentiation of DNA damage during oxidative stress. The second specific aim is to test the hypothesis that NO inhibits the mitochondrial pathway of apoptosis by directly nitrosylating procaspase 9 rendering cholangiocarcinoma cells resistant to apoptosis-inducing treatments. The third specific aim is to test the hypothesis that iNOS expression enhances tumor progression and metastases in cholangiocarcinoma xenografts and promotes the development and progression of cholangiocarcinoma in an animal model of this disease. Successful completion of these proposed studies has the potential for development of new strategies to prevent and treat malignant human liver diseases.